8.1
Nucleic Acid Stains
Molecular Probes prepares the most extensive assortment of nucleic acid stains commercially available, many of which have been developed in our research laboratories.
This section discusses the physical properties of the various classes of dyes, listed below.
The sections in Chapter 8 that follow discuss numerous applications of these dyes and
our other reagents and technology for genomics research.
The four classes of Molecular Probes’ proprietary cyanine dyes include:
•
•
•
•
Premiere dyes for ultrasensitive solution quantitation and gel staining (Table 8.1)
The cell-impermeant TOTO, TO-PRO and SYTOX families of dyes (Table 8.2)
The cell-permeant SYTO family of dyes (Table 8.3)
Chemically reactive SYBR dyes that can be used to form bioconjugates (see below)
The three classes of classic nucleic acid stains (Table 8.4) include:
• Intercalating dyes, such as ethidium bromide and propidium iodide
• Minor-groove binders, such as DAPI and the Hoechst dyes
• Miscellaneous nucleic acid stains, including acridine orange, 7-AAD, LDS 751 and
hydroxystilbamidine, with special properties
Properties of Cyanine Dyes
Over the years, Molecular Probes’ researchers have invented many nucleic acid–
binding cyanine dye derivatives that share several unique and outstanding properties:
Figure 8.1 Normalized fluorescence emission
spectra of DNA-bound cyanine dimers, identified
by the color key on the sidebar.
• High molar absorptivity, with extinction coefficients typically greater than
50,000 cm-1M-1 at visible wavelengths
• Very low intrinsic fluorescence, with quantum yields usually less than 0.01 when
not bound to nucleic acids
• Large fluorescence enhancements (often over 1000-fold) upon binding to nucleic
acids, with increases in quantum yields to as high as 0.9
• Moderate to very high affinity for nucleic acids, with little or no staining of other
biopolymers
Representatives of this class of nucleic acid stains have fluorescence excitations and
emissions that span the visible-light spectrum from blue to near infrared (Figure 8.1) with
additional absorption peaks in the UV, making them compatible with many different
types of instrumentation. The cyanine dyes show differences in some physical characteristics — particularly differences in permeability to cell membranes and nucleic acid
specificity — that allow their distribution into distinct classes. Those classes are discussed in detail in the following sections of this chapter.
Premiere Cyanine Dyes for Ultrasensitive Nucleic Acid Detection
and Quantitation
Several of our cyanine dyes give superior results in specific assays for the analysis of
nucleic acids (Table 8.1). For these dyes, we have developed detailed and extensively
tested protocols to facilitate reproducible, high-sensitivity results in these assays.
• The PicoGreen, OliGreen and RiboGreen quantitation reagents in Section 8.3 set a
benchmark for the detection and quantitation of DNA, RNA and oligonucleotides in
solution. These reagents offer extremely simple and rapid protocols as well as linear
ranges that span up to four orders of magnitude in nucleic acid concentration.
• The SYBR Gold, SYBR Green I and SYBR Green II nucleic acid gel stains in Section
8.4 are ultrasensitive gel stains that surpass the sensitivity of ethidium bromide by
more than an order of magnitude in nucleic acid detection. Furthermore, Ames testing
by an independent laboratory has shown that the SYBR Green I stain is significantly
less mutagenic than ethidium bromide 1 (Figure 8.62).
A space-filling model of DNA.
Section 8.1
269
• SYBR DX DNA blot stain (S-7550, Section 8.5) allows the
direct detection of DNA on filter membranes after Southern
transfer, with sensitivity equivalent to that achieved with
silver-enhanced gold staining.
• The CyQUANT GR dye (C-7026) in Section 15.4 is a
reagent for quantitating cell proliferation that can reliably
detect the nucleic acids in as few as 50 cells.
Cell-Impermeant Cyanine Dimers:
The TOTO Family of Dyes
The patented cyanine dimer dyes listed in Table 8.2 are often
referred to as the TOTO family of dyes. These dyes are symmetric dimers of cyanine dyes with exceptional sensitivity for nucleic acids.2 This sensitivity is due to a high affinity for nucleic
acids, in combination with a very high fluorescence enhancement and quantum yield upon binding. The unique physical
characteristics of these dyes and some illustrative applications
are discussed below. Specific applications are discussed in later
sections of this chapter.
Each of the cyanine dimer dyes is available separately (Table
8.2). For researchers designing new applications, the Nucleic
Acid Stains Dimer Sampler Kit (N-7565) provides samples of
eight spectrally distinct analogs of the dimeric cyanine dyes for
testing (Table 8.2).
High Affinity for Nucleic Acids
Appropriately designed dimers of nucleic acid–binding dyes
have nucleic acid–binding affinities that are several orders of
magnitude greater than those of their parent compounds.3–5 For
example, the intrinsic DNA binding affinity constants of ethidium
bromide (E-1305, E-3565) and ethidium homodimer-1 (E-1169)
are reported to be 1.5 × 105 and 2 × 108 M-1, respectively, in
0.2 M Na+.6 As a result, the dimeric cyanine dyes are among the
highest-affinity fluorescent probes available for nucleic acid
staining. For example, in the TOTO-1 dimeric cyanine dye
(T-3600), the positively charged side chains of the TO-PRO-1
monomeric cyanine dye (T-3602, Figure 8.2) are covalently
linked to form the TOTO-1 molecule, with four positive charges
(Figure 8.3). This linkage gives the TOTO-1 dye a greatly enhanced affinity for nucleic acids — over 100 times greater than
that of the TO-PRO-1 monomer. The TOTO-1 dye exhibits a
higher affinity for double-stranded DNA (dsDNA) than even the
ethidium homodimers and also binds to both single-stranded
DNA (ssDNA) and RNA. The extraordinary stability of TOTO-1–
nucleic acid complexes 4,7,8 ensures that the dye–DNA association
remains stable, even during electrophoresis (Figure 8.4); thus,
samples can be prestained with nanomolar dye concentrations
prior to electrophoresis,9,10 thereby reducing the hazards inherent
in handling large volumes of ethidium bromide staining solutions.4,8,11 In contrast, the binding of thiazole orange — the parent
Table 8.1 Specialty nucleic acid reagents for molecular biology.
Cat #
Dye Name
Ex/Em *
Application
Dyes for Ultrasensitive Solution Quantitation
P-7581, P-11495,
P-7589, P-11496,
R-21495, R-21496
PicoGreen Quantitation
Reagent and Kits
502/523
Ultrasensitive reagent for solution quantitation of dsDNA
O-7582, O-11492
OliGreen Quantitation
Reagent and Kit
498/518
Ultrasensitive reagent for solution quantitation of ssDNA and oligonucleotides
R-11491, R-11490
RiboGreen Quantitation
Reagent and Kit
500/520
Ultrasensitive reagent for solution quantitation of RNA
Dyes for Sensitive Detection of Nucleic Acids in Gels and on Blots
S-11494
SYBR Gold stain
495/537
Ultrasensitive gel stain for single- or double-stranded DNA or RNA post-electrophoresis
S-7567, S-7563,
S-7585
SYBR Green I stain
494/521
S-7568, S-7564,
S-7586
SYBR Green II stain
492/513
Sensitive stain for RNA and ssDNA post-electrophoresis
S-7550
SYBR DX DNA blot stain
475/499
Sensitive stain for DNA on blots (not recommended for staining RNA)
Ultrasensitive gel stain for double-stranded DNA and oligonucleotides post-electrophoresis
Also useful for real-time PCR assays
* All excitation (Ex) and emission (Em) maxima, in nm, were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution.
Figure 8.2 T-3602 TO-PRO-1 iodide (515/531).
270
Figure 8.3 T-3600 TOTO-1 iodide (514/533).
Chapter 8 — Nucleic Acid Detection and Genomics Technology
www.probes.com
compound of TOTO-1 and TO-PRO-1 — is rapidly reversible, limiting the dye’s sensitivity and rendering its nucleic acid complex unstable to electrophoresis.11
High Fluorescence Enhancements and High Quantum Yields upon Binding to
Nucleic Acids
In addition to their superior binding properties, TOTO-1 dye and the other cyanine
dimers are essentially nonfluorescent in the absence of nucleic acids and exhibit significant fluorescence enhancements upon DNA binding 7,12 (100- to 1000-fold), which comTable 8.2 Cell membrane–impermeant cyanine nucleic acid stains.
Figure 8.4 Lambda bacteriophage Hin dIII fragments were prestained with various nucleic acid
dyes, run on a 0.7% agarose gel and visualized using a standard 300 nm UV transilluminator. From
left to right, the dyes used were: POPO-1 (P-3580),
BOBO-1 (B-3582), YOYO-1 (Y-3601), TOTO-1
(T-3600), JOJO-1 (J-11372), POPO-3 (P-3584),
LOLO-1 (L-11376), BOBO-3 (B-3586), YOYO-3
(Y-3606) and TOTO-3 (T-3604) nucleic acid stains.
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Section 8.1
271
pares favorably with the fluorescence enhancement of thiazole orange upon DNA
binding 13 (~3000-fold). Furthermore, the fluorescence quantum yields of the cyanine
dimers bound to DNA are high (generally between 0.2 and 0.6), and their extinction
coefficients are an order of magnitude greater than those of the ethidium homodimers.7
Their sensitivity is sufficient for detecting single molecules of labeled nucleic acids by
optical imaging (Figure 8.5) and flow cytometry (Section 8.4) and for tracking dyelabeled virus particles in microbial communities and aquatic systems by fluorescence
microscopy.14,15 These dyes are generally considered to be cell impermeant, although
their use to stain reticulocytes permeabilized by 5% DMSO has been reported.16,17
Figure 8.5 The relaxation of a single, 39 µm–
long DNA molecule stained with YOYO-1 iodide
(Y-3601) imaged at 4.5 second intervals. After the
1 µm polystyrene sphere was trapped with optical
tweezers, the attached DNA was stretched to its
full extension in a fluid flow and then allowed to relax upon stoppage of fluid flow due to its entropic
elasticity (Science 264, 822 (1994)). The YOYO-1
iodide–DNA complex is excited with the 488 nm
spectral line of the argon-ion laser and visualized
through a 515 nm longpass optical filter using a
Hamamatsu SIT camera with image processing.
Image contributed by Thomas Perkins, Department
of Physics, Stanford University.
Figure 8.6 NMR solution structure of the TOTO-1
dye (T-3600) bound to DNA. The NMR structure
shows that TOTO-1 binds to DNA through bis-intercalation. The image was derived from data submitted to the Protein Data Bank (www.rcsb.org/
pdb/, (Nucleic Acids Res 28, 235 (2000))), number
PDB 108D. The initial structure was described in
Biochemistry 34, 8542 (1995).
272
Modifying the Dimers Creates Compounds with Different Spectral Characteristics
Simply by changing the aromatic rings and the number of carbon atoms linking the
cyanine monomers, we were able to synthesize an extended series of these dyes with
different spectral characteristics. Chemical modifications produce dramatic shifts in the
molecules’ absorption and emission spectra and reduce the quantum yields of the bound
dyes but cause little or no change in their high affinity for DNA. The names of the dyes
reflect their basic structure and spectral characteristics. For example, YOYO-1 iodide
(491/509) has one carbon atom bridging the aromatic rings of the oxacyanine dye and
exhibits absorption/emission maxima of 491/509 nm when bound to dsDNA. The dsDNA
complex of the YOYO-3 dye (612/631) — which differs from the YOYO-1 dye only in
the number of bridging carbon atoms (three) — has absorption/emission maxima of
612/631 nm when bound to dsDNA. Fluorescence spectra for the POPO, BOBO, YOYO,
TOTO, JOJO and LOLO dyes bound to dsDNA are shown in Figure 8.1. The spectra of
these dyes at dye:base ratios of less than 1:1 are essentially the same for the corresponding dye–ssDNA and dye–RNA complexes. At higher dye:base ratios, however, ssDNA
and RNA complexes of all of the monomethine (“-1”) dyes of the TOTO series and
TO-PRO series have red-shifted emissions, whereas corresponding complexes of the
trimethine (“-3”) analogs do not. Thus, the cyanine dimer family provides dyes with a
broad range of spectral characteristics to match the peak excitation light of almost any
available light source. Some common light sources that match each dye are shown in
Table 8.2.
Binding Modes of the Cyanine Dimers
The studies on cyanine dimer binding modes have focused on the YOYO-1 and
TOTO-1 dyes. The YOYO-1 dye was found to exhibit at least two distinct binding
modes. At low dye:base pair ratios, the binding mode appears to consist primarily of bisintercalation.18–20 Each monomer unit intercalates between bases, with the benzazolium
ring system sandwiched between the pyrimidines and the quinolinium ring between the
purine rings, causing the helix to unwind.20 The distortion in the local DNA structure
caused by YOYO-1 bis-intercalation has been observed by two-dimensional NMR spectroscopy.21 At high dye:base pair ratios, a second, less well characterized mode of external binding begins to contribute.18,19 Circular dichroism measurements also indicate a
possible difference in the binding modes of the YOYO-1 dye to ssDNA and dsDNA.22
These data are consistent with our own results, including the observation that the
fluorescence emission of the YOYO-1 dye complex with nucleic acids shifts to longer
wavelengths at high dye:base ratios upon binding to single-stranded nucleic acids and
that the salt, ethanol and sodium dodecyl sulfate (SDS) sensitivity of YOYO-1 dye binding to DNA is a function of the dye:base pair ratio.23
The TOTO-1 dye is capable of bis-intercalation,24 although it reportedly interacts with
dsDNA and ssDNA with similarly high affinity.3 NMR studies of TOTO-1 dye interactions
with a double-stranded 8-mer indicate that TOTO-1 dye is a bis-intercalator, with the fluorophores intercalating between the bases and the linker region having interactions in the
minor groove 25 (Figure 8.6). Binding of the dye partially unwinds the DNA,25 distorting
and elongating the helix.26 However, another study using fluorescence polarization measurements suggests that an external binding mode, where the dipole of the dye molecules
is aligned with the DNA grooves, may be more important.27 The TOTO-1 dye reportedly
exhibits some sequence selectivity for the site 5′-CTAG-3′, although it will bind to almost
any sequence in dsDNA.28–31 The TOTO-1 dye does not exhibit cooperative binding to
DNA, suggesting that it will be a suitable dye for detecting nucleic acids in gels.29
Chapter 8 — Nucleic Acid Detection and Genomics Technology
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The binding modes of the other members of the TOTO dye
series have also been partially characterized. Electrophoresis and
fluorescence lifetime measurements have shown that the YOYO-3
dye also appears to intercalate into DNA.32 During application
development, we have determined that staining of nucleic acids
by the BOBO-1 and POPO-1 dyes is much faster (occurring
within minutes) than staining by the YOYO-1 or TOTO-1 dyes
(which can take several hours to reach equilibrium under the
same experimental conditions),24 indicating possible differences
in their binding mechanisms. Fluorescence yield and lifetime
measurements have been used to assess the base selectivity of an
extensive series of these dyes.12 Circular dichroism measurements
have shown that bis-intercalation is the predominant binding
mode for the POPO-1 dye.33
Working with Cyanine Dimers
All of the dyes in the TOTO series (Table 8.2) are supplied
as 1 mM solutions in dimethylsulfoxide (DMSO), except for
POPO-3 (P-3584), which is supplied as a 1 mM solution in dimethylformamide (DMF). These cationic dyes appear to be readily adsorbed out of aqueous solutions onto surfaces (particularly
glass) but are very stable once complexed to nucleic acids. Several applications of these dyes for staining nucleic acids in solutions, gels, microarrays and cells are described in Section 8.3,
Section 8.5, Section 8.6, Section 8.7 and Section 15.5.
Live Cell–Impermeant Cyanine Monomers:
The TO-PRO Family of Dyes
Our patented TO-PRO family of dyes, all of which are listed
in Table 8.2, each comprise a single cyanine dye and a cationic
side chain (Figure 8.2). The eleven dyes in the TO-PRO series
are spectrally analogous to the corresponding dimeric cyanine
dyes; however, with only two positive charges and one intercalating unit, the TO-PRO dyes exhibit somewhat reduced affinity for
nucleic acids relative to the dyes in the TOTO series.34 Like their
dimeric counterparts, these monomeric cyanine dyes are typically
impermeant to cells,35 although the YO-PRO-1 (Y-3603) dye has
been shown to be permeant to apoptotic cells, providing a convenient indicator of apoptosis 36–39 (Section 15.5, Figure 15.67) and
to pass through P2X7 receptor channels of live cells.40–42
Spectral Characteristics of the Cyanine Dye Monomers
The TO-PRO family of dyes retains all of the exceptional
spectral properties of the dimeric cyanine dyes discussed above.
The absorption and emission spectra of these monomeric cyanine
dyes cover the visible and near-IR spectrum (Table 8.2). They
also have relatively narrow emission bandwidths, thus facilitating multicolor applications in imaging and flow cytometry. The
YO-PRO-1 (491/509) and TO-PRO-1 (515/531) dyes are optimally excited by the 488 nm and 514 nm spectral lines of the argonion laser, respectively. In flow cytometric analysis, the TO-PRO-3
(642/661) complex with nucleic acids has been excited directly
by the red He–Ne laser 43 and indirectly by the argon-ion laser by
using fluorescence resonance energy transfer (FRET, see Section
1.3) from co-bound propidium iodide.44 The TO-PRO-3 complex
with nucleic acids has also been detected in a flow cytometer
equipped with an inexpensive 3 mW visible-wavelength diode
laser that provides excitation at 635 nm.45 Although the DNAinduced fluorescence enhancement of the TO-PRO-5 dye (T-7596)
is not as large as that observed with our other cyanine dyes, its
spectral characteristics (excitation/emission maxima ~745/770 nm)
provide a unique alternative for multicolor applications and specialized instrumentation.
Binding of Cyanine Dye Monomers to Nucleic Acids
The binding affinity of the TO-PRO series of dyes to dsDNA
is lower than that of the TOTO series of dyes but is still very
high, with dissociation constants in the micromolar range.46
TO-PRO dyes also bind to RNA and ssDNA, although typically
with somewhat lower fluorescence quantum yields. Fluorescence
polarization studies indicate that the TO-PRO-1 and PO-PRO-1
dyes bind by intercalation, with unwinding angles of 2° and 31°,
respectively.33 Binding of these dyes to dsDNA is not sequence
selective.47
Working with Cyanine Monomers
All dyes of the TO-PRO series (Table 8.2) are supplied as
1 mM solutions in DMSO. Various applications of the TO-PRO
series of dyes for staining nucleic acids are described in Section
8.3, Section 8.5, Section 8.6 and Section 15.5.
Cell-Impermeant SYTOX Dyes for
Dead-Cell Staining
Our three SYTOX nucleic acid stains (Table 8.2) are cellimpermeant cyanine dyes that are particularly good dead-cell
stains.
SYTOX Green Stain
The SYTOX Green nucleic acid stain (S-7020) is a highaffinity nucleic acid stain that easily penetrates cells that have
compromised plasma membranes and yet will not cross the membranes of live cells. It is especially useful for staining both grampositive and gram-negative bacteria — and probably virus particles 15,48 — where an exceptionally bright signal is required.
Following brief incubation with the SYTOX Green stain, the
nucleic acids of dead cells fluoresce bright green when excited
with the 488 nm spectral line of the argon-ion laser or with any
other 450–500 nm source. No wash steps are required since all
of the SYTOX dyes are essentially nonfluorescent in aqueous
medium. Unlike the DAPI or Hoechst dyes, the SYTOX Green
nucleic acid stain shows little base selectivity. These properties,
combined with its ~1000-fold fluorescence enhancement upon
nucleic acid binding and high quantum yield, make our SYTOX
Green stain a simple and quantitative single-step dead-cell indicator for use with epifluorescence and confocal laser-scanning
microscopes, fluorometers, fluorescence microplate readers and
flow cytometers (Figure 15.11). The SYTOX Green dye is included as a dead-cell stain in our Vybrant Apoptosis Assay Kit #1
(V-13240, Section 15.5) and in our ViaGram Red+ Bacterial
Gram Stain and Viability Kit (V-7023, Section 15.3).
The SYTOX Green nucleic acid stain may be used with blueand red-fluorescent labels for multiparameter analyses (Figure
8.7). It is also possible to combine the SYTOX Green nucleic
acid stain with the SYTO 17 red-fluorescent nucleic acid stain
Section 8.1
273
(S-7579) for two-color visualization of dead and live cells (Section 15.3). Because the
SYTOX Green nucleic acid stain is an excellent DNA counterstain for chromosome
labeling and for fixed cells and tissues (Figure 8.8), we have incorporated it into our
Cytological Nuclear Counterstain Kit (C-7590), which is discussed in Section 8.6.
The SYTOX Green stain is also used in our Vybrant Tumor Necrosis Factor (TNF)
Assay Kit 49 (V-23100, Section 15.3).
Figure 8.7 Bovine pulmonary artery endothelial
cells (BPAEC) incubated with the fixable, mitochondrion-selective MitoTracker Red CMXRos
(M-7512). After staining, the cells were formaldehyde-fixed, acetone-permeabilized, treated with
DNase-free RNase and counterstained using
SYTOX Green nucleic acid stain (S-7020) from our
Cytological Nuclear Counterstain Kit (C-7590). Microtubules were labeled with a mouse monoclonal
anti–β-tubulin antibody, biotin-XX goat anti–mouse
IgG antibody (B-2763) and Cascade Blue NeutrAvidin biotin-binding protein (A-2663). This photograph was taken using multiple exposures through
bandpass optical filters appropriate for Texas Red
dye, fluorescein and DAPI using a Nikon Labophot
2 microscope equipped with a Quadfluor epi-illumination system.
SYTOX Blue Stain
Like the SYTOX Green nucleic acid stain (S-7020), our SYTOX Blue stain (S-11348)
is a high-affinity nucleic acid stain that typically penetrates only those cells that have
compromised plasma membranes (Figure 8.9). The SYTOX Blue stain labels both DNA
and RNA with extremely bright fluorescence centered near 480 nm (Figure 8.106). The
absorption maximum of the nucleic acid–bound SYTOX Blue stain (~431 nm) permits
very efficient fluorescence excitation by the 436 nm spectral line of the mercury-arc
lamp. Unlike many blue-fluorescent dyes, the SYTOX Blue stain is also efficiently excited by tungsten–halogen lamps and other sources that have relatively poor emission in the
UV portion of the spectrum. The brightness of the SYTOX Blue stain allows sensitive
detection with fluorometers, microplate readers, arc-lamp–equipped flow cytometers and
epifluorescence microscopes, including those not equipped with UV-pass optics.
In a side-by-side comparison with the SYTOX Green stain, the SYTOX Blue stain
yielded identical results when quantitating membrane-compromised bacterial cells. And
like the SYTOX Green stain, the SYTOX Blue stain does not interfere with bacterial cell
growth. Because their emission spectra overlap somewhat, we have found that it is not
ideal to use the SYTOX Blue stain and green-fluorescent dyes together; however, fluorescence emission of the SYTOX Blue stain permits clear discrimination from orange- or
red-fluorescent probes, facilitating the development of multicolor assays with minimal
spectral overlap between signals.
SYTOX Orange Stain
Our SYTOX Orange nucleic acid stain (S-11368) clearly distinguishes dead bacteria,
yeast or mammalian cells. The SYTOX Orange stain has shorter-wavelength emission
compared to propidium iodide and matches the rhodamine filter set more closely (Figure
8.10). In addition, the SYTOX Orange stain has a much higher molar absorptivity (extinction coefficient) than propidium iodide and the SYTOX Orange stain has a far greater
fluorescence enhancement upon binding DNA, suggesting that it may have a higher
sensitivity as a dead-cell stain or as a nuclear counterstain. The SYTOX Orange stain
was shown to be the best dye for DNA fragment sizing by single-molecule flow cytometry when using a Nd:YAG excitation source, with a 450-fold enhancement on binding to
dsDNA.50
Cell-Permeant Cyanine Dyes: The SYTO Nucleic Acid Stains
Figure 8.8 Adult zebrafish gut cryosections that
have been incubated with BODIPY TR-X phallacidin
(B-7464), followed by the SYTOX Green nucleic
acid stain (S-7020), and then dehydrated and
mounted. The image was obtained by taking multiple exposures through bandpass optical filter sets
appropriate for fluorescein and the Texas Red dye.
The use of nucleic acid stains for
assessing cell viability and cytotoxicity
is described in Section 15.2.
274
The numerous patented SYTO dyes in Table 8.3 are lower-affinity nucleic acid stains
that passively diffuse through the membranes of most cells.51 These UV- or visible light–
excitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells,
as well as in gram-positive and gram-negative bacteria. Molecular Probes has synthesized
a large number of SYTO dyes (Table 8.3) that share several important characteristics:
• Permeability to virtually all cell membranes, including mammalian cells and bacteria
(Chapter 15)
• High molar absorptivity, with extinction coefficients greater than 50,000 cm-1M-1 at
visible absorption maxima
• Extremely low intrinsic fluorescence, with quantum yields typically less than 0.01
when not bound to nucleic acids
• Quantum yields typically greater than 0.4 when bound to nucleic acids
Available as blue-, green-, orange- or red-fluorescent dyes, these novel SYTO stains
provide researchers with visible light–excitable dyes for labeling DNA and RNA in live
cells (Figure 8.11). The SYTO dyes may also be useful for nucleic acid detection in
Chapter 8 — Nucleic Acid Detection and Genomics Technology
www.probes.com
solution, in electrophoretic gels, on blots, on microarrays and in several other assays.
SYTO dyes differ from each other in one or more characteristics, including cell permeability, fluorescence enhancement upon binding nucleic acids, excitation and emission
spectra (Table 8.3), DNA/RNA selectivity and binding affinity. The SYTO dyes are
compatible with a variety of fluorescence-based instruments that use either laser excitation or a conventional broadband illumination source (e.g., mercury- and xenon-arc
lamps).
The SYTO dyes can stain both DNA and RNA. In most cases, the fluorescence wavelengths and emission intensities are similar for solution measurements of DNA or RNA
binding. Exceptions that we know of include the SYTO 12 and SYTO 14 dyes, which are
about twice as fluorescent on RNA than DNA, and SYTO 16, which is about twice as
fluorescent on DNA than RNA. Consequently, the SYTO dyes do not act exclusively as
nuclear stains in live cells and should not be equated in this regard with compounds such
as DAPI or the Hoechst 33258 and Hoechst 33342 dyes, which, because of their DNA
selectivity, readily stain cell nuclei at low concentrations in most cells. SYTO dye–
stained eukaryotic cells will generally show diffuse cytoplasmic staining, as well as
nuclear staining. The SYTO 14 dye (S-7576) has been used to visualize the translocation
of endogenous RNA found in polyribosome complexes in living cells.52,53 Particularly
intense staining of intranuclear bodies is frequently observed. Because these dyes are
generally cell permeant and most of the SYTO dyes contain a net positive charge at
neutral pH, they may also stain mitochondria. In addition, the SYTO dyes will stain most
gram-positive and gram-negative bacterial cells. Dead yeast cells are brightly stained
with the SYTO dyes, and live yeast cells typically exhibit staining of both the mitochondria and the nucleus. Some of the SYTO dyes have been reported to be useful for detecting apoptosis 54,55 (Section 15.5), and dyes structurally similar to the SYTO dyes have
been used to detect multidrug-resistant cells 56 (Section 15.6). The red-fluorescent SYTO
dyes are proving useful as counterstains (Section 8.6) when combined with green-fluorescent antibodies (Section 7.3), lectins (Section 7.7) or the cell-impermeant SYTOX
Green nucleic acid stain (see above). Several of the green-fluorescent SYTO dyes are
excellent nuclear counterstains. We anticipate that many more applications will be found
for these unique nucleic acid stains.
All of our patented SYTO dyes are available separately, with the exception of the
green-fluorescent SYTO 9 and SYTO 10 dyes, which are used in some of our LIVE/
DEAD Kits (Section 15.3, Table 15.2) and the SYTO BC reagent, which is used in our
Bacteria Counting Kit (B-7277, Section 15.4). The green-fluorescent SYBR 14 dye, a
component of our LIVE/DEAD Sperm Viability Kit (L-7011, Section 15.3) is also in the
Figure 8.9 A mixed population of live and isopropyl alcohol–killed Micrococcus luteus stained with
SYTOX Blue nucleic acid stain (S-11348), which
does not penetrate intact plasma membranes.
Dead cells exhibit bright blue-fluorescent staining.
The image was acquired using a longpass optical
filter set appropriate for the Cascade Blue dye.
Figure 8.10 Absorption and fluorescence emission
spectra of SYTOX Orange nucleic acid stain
(S-11368) bound to DNA.
Our four SYTO dye sampler kits provide a total of 24 membrane-permeant
nucleic acid stains. The kits are ideal
for screening purposes.
Figure 8.11 Human neutrophil nuclei stained with SYTO 13 live-cell nucleic acid stain (S-7575).
The photo was acquired using an optical filter appropriate for fluorescein and differential interference contrast (DIC) sequentially in a Nikon Eclipse E800 microscope.
Section 8.1
275
Table 8.3 Cell-permeant cyanine nucleic acid stains.*
Figure 8.12 E-1305 ethidium bromide.
Figure 8.13 P-1304 propidium iodide.
Figure 8.14 Day 10 of development of a Drosophila
ovarian egg chamber assembly line. The nuclei of
follicle and nurse cells were labeled with propidium
iodide (P-1304, P-3566, P-21493) and visualized by
confocal laser-scanning microscopy using excitation
by the 568 nm spectral line of an Ar–Kr laser. Image
contributed by Sandra Orsulic, Department of Biology, University of North Carolina at Chapel Hill.
Figure 8.15 Normalized fluorescence emission
spectra of DNA-bound 1) Hoechst 33258 (H-1398,
H-3569, H-21491), 2) acridine orange (A-1301,
A-3568), 3) ethidium bromide (E-1305, E-3565)
and 4) 7-aminoactinomycin D (A-1310).
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SYTO family of dyes. To facilitate testing the SYTO dyes in new
applications, we also offer several sampler kits containing sample
sizes of SYTO dyes in each color set (Table 8.3). With each
purchase of a sampler kit or individual reagent, we include a
detailed product information sheet, describing the spectral properties of the dyes, to assist the researcher in designing staining
protocols. The recommended dye concentration for cell staining
depends on the assay and may vary widely but is typically
1–20 µM for bacteria, 1–100 µM for yeast and 10 nM–5 µM
for other eukaryotes.
Chemically Reactive Cyanine Dyes
The amine-reactive succinimidyl esters of the SYBR 101,
SYBR 102 and SYBR 103 dyes (S-21500, S-21501, S-21502)
can be conjugated to peptides, proteins, drugs, polymeric matrices and biomolecules with primary amine groups. The conjugates
are expected to be essentially nonfluorescent until they are able to
complex with nucleic acids, resulting in strong green fluorescence. Thus, they may be useful for studies of nucleic acid binding to various biomolecules, such as DNA-binding proteins. It is
also possible that conjugates of other biomolecules may be capable of monitoring their transport into the nucleus. SYBR dye–
conjugates of solid or semisolid matrices (such as microspheres,
magnetic particles or various resins) may be useful for detection
or affinity isolation of nucleic acids.
The reactive SYBR dyes may also be conjugated to aminemodified nucleic acids. Although it is possible that the SYBR
dyes may show fluorescence when conjugated to amine groups on
nucleic acids, they may be useful for developing homogeneous
hybridization assays, in which a specific sequence can be quantitated in solution without the need to separate bound and free
probes. For example, a similar reactive nucleic acid stain has been
used to label peptide nucleic acids (PNAs) for use as probes in
real-time PCR. The labeled PNA probes exhibited a fluorescence
increase upon hybridization to their complementary sequence and
the ability to identify a single-base mismatch in a ten-base target
sequence.57,58
Phenanthridines and Acridines:
Classic Intercalating Dyes
Cell-Impermeant Ethidium Bromide and Propidium Iodide
Ethidium bromide (EtBr, E-1305; E-3565; Figure 8.12)
and propidium iodide (PI, P-1304; P-3566; FluoroPure grade,
P-21493; Figure 8.13) are structurally similar phenanthridinium
intercalators. PI is more soluble in water and less membranepermeant than EtBr, although both dyes are generally excluded
from viable cells. EtBr and PI can be excited with mercury- or
xenon-arc lamps or with the argon-ion laser, making them suitable for fluorescence microscopy, confocal laser-scanning microscopy (Figure 8.14), flow cytometry and fluorometry. These
dyes bind with little or no sequence preference at a stoichiometry
of one dye per 4–5 base pairs of DNA.59 Excitation of the EtBr–
DNA complex may result in photobleaching of the dye and single-strand breaks.60 Both EtBr and PI also bind to RNA, necessitating treatment with nucleases to distinguish between RNA and
DNA. Once these dyes are bound to nucleic acids, their fluores-
cence is enhanced ~20- to 30-fold, their excitation maxima are
shifted ~30–40 nm to the red and their emission maxima are
shifted ~15 nm to the blue 61 (Figure 8.15, Table 8.4). Although
their molar absorptivities (extinction coefficients) are relatively
low, EtBr and PI exhibit sufficiently large Stokes shifts to allow
simultaneous detection of nuclear DNA and fluorescein-labeled
antibodies, provided that the proper optical filters are used
(Table 24.8).
PI is commonly used as a nuclear or chromosome counterstain
(Section 8.6, Figure 8.14) and as a stain for dead cells (Section
15.2, Figure 15.17). EtBr currently is the most commonly used
general nucleic acid gel stain (Section 8.4). However, our SYBR
Gold and SYBR Green nucleic acid gel stains are far more sensitive than EtBr, and the SYBR Green I stain has been shown to be
significantly less mutagenic than EtBr by Ames testing 1 (Section
8.4, Figure 8.62). EtBr and PI are potent mutagens and must be
handled with extreme care. Solutions containing EtBr or PI can
be decontaminated by filtration through activated charcoal, which
is then incinerated, thus providing an economical decontamination
procedure.62 Alternatively, the dyes can be completely degraded
in buffer by reaction with sodium nitrite and hypophosphorous
acid.63 EtBr and PI are offered as solids (E-1305, P-1304; FluoroPure grade, P-21493) as well as in aqueous solution (E-3565,
P-3566), enabling researchers to avoid contact with the mutagenic
powders.
Cell-Permeant Hexidium Iodide
Molecular Probes’ patented hexidium iodide reagent (H-7593)
is a moderately lipophilic phenanthridinium dye (Figure 8.16)
that is permeant to mammalian cells and selectively stains almost
all gram-positive bacteria in the presence of gram-negative bacteria.64 Our LIVE BacLight Bacterial Gram Stain Kit and ViaGram
Red+ Bacterial Gram Stain and Viability Kit (L-7005, V-7023;
Section 15.3) use hexidium iodide for the discrimination of bacterial gram sign (Figure 15.42). Hexidium iodide yields slightly
shorter-wavelength spectra upon DNA binding than our ethidium
or propidium dyes. Generally, both the cytoplasm and nuclei of
eukaryotic cells show staining with hexidium iodide; however,
mitochondria and nucleoli can also be stained.
Cell-Permeant Dihydroethidium (Hydroethidine)
Dihydroethidium (also known as hydroethidine) is a chemically reduced ethidium derivative (Figure 15.20) that is permeant to
live cells. Dihydroethidium exhibits blue fluorescence in the
cytoplasm. Many viable cells oxidize the probe to ethidium,
which then fluoresces red upon DNA intercalation 65–67 (Figure
19.12). Dihydroethidium, which is somewhat air sensitive, is
available in a 25 mg vial (D-1168) or specially packaged in 10
vials of 1 mg each (D-11347); the special packaging is strongly
recommended when small quantities of the dye will be used at a
time. Dihydroethidium is also available as a 5 mM stabilized
solution in dimethylsulfoxide (D-23107).
High-Affinity Ethidium Homodimers
Ethidium homodimer-1 (EthD-1, E-1169; Figure 8.17) and
ethidium homodimer-2 (EthD-2, E-3599; Figure 8.18) strongly
bind to dsDNA, ssDNA, RNA and oligonucleotides with a large
fluorescence enhancement (>30-fold). EthD-1 also binds with
high affinity to triplex nucleic acid structures.68 One molecule of
Section 8.1
277
Table 8.4 Properties of classic nucleic acid stains.
Cat #
Dye Name
Ex/Em *
Applications †
Fluorescence
Emission
Color
Green
• Impermeant
• High-affinity DNA binding
• AT-selective
A-666
Acridine homodimer
431/498
A-1301
A-3568 ‡
Acridine orange
500/526 (DNA)
460/650 (RNA)
A-1310
7-AAD (7-aminoactinomycin D)
546/647
Red
• Weakly permeant
• GC-selective
A-7592
Actinomycin D
442
None
• Chromosome banding
A-1324
ACMA
419/483
Blue
• AT-selective
• Alternative to quinacrine for chromosome Q banding
• Membrane phenomena
D-1306
D-3571
D-21490
DAPI
358/461
Blue
• Semi-permeant
• AT-selective
• Cell-cycle studies
D-1168
D-11347
D-23107
Dihydroethidium
518/605
Red §
• Permeant
• Blue fluorescent until oxidized to ethidium
E-1305
E-3565 ‡
Ethidium bromide
518/605
Red
•
•
•
•
E-1169
Ethidium homodimer-1
(EthD-1)
528/617
Red
• Impermeant
• Dead-cell stain
• High-affinity DNA labeling
• Electrophoresis prestain
• Argon-ion and green He–Ne laser excitable
E-3599
Ethidium homodimer-2
(EthD-2)
535/624
Red
• Impermeant
• Very high-affinity DNA labeling
• Electrophoresis prestain
E-1374
Ethidium monoazide
464/625
(unbound)**
Red
• Impermeant
• Photocrosslinkable
• Compatible with fixation procedures
H-7593
Hexidium iodide
518/600
Red
• Permeant, except gram-negative bacteria
• Stains nuclei and cytoplasm of eukaryotes and some bacteria
H-1398
H-3569 ‡
H-21491
Hoechst 33258
(bis-benzimide)
352/461
Blue
• Permeant
• AT-selective
• Minor groove–binding
• dsDNA-selective binding
• Cell-cycle studies
• Chromosome and nuclear counterstain
H-1399
H-3570 ‡
H-21492
Hoechst 33342
350/461
Blue
• Permeant
• AT-selective
• Minor groove–binding
• dsDNA-selective binding
• Cell-cycle studies
• Chromosome and nuclear counterstain
H-21486
Hoechst 34580
392/498
Blue
• Permeant
• AT-selective
• Minor groove–binding
• dsDNA-selective binding
• Cell-cycle studies
• Chromosome and nuclear counterstain
H-22845
Hydroxystilbamidine
385/emission
varies with
nucleic acid
L-7595
LDS 751
543/712 (DNA)
590/607 (RNA)
N-21485
Nuclear yellow
355/495
Yellow
P-1304
P-3566 ‡
P-21493
Propidium iodide (PI)
535/617
Red
Green/Red
Varies
Red/infrared
• Permeant
• Lysosome labeling
• Flow cytometry
• Cell-cycle studies
• RNA/DNA discrimination measurements
•
•
•
•
Impermeant
dsDNA intercalator
Dead-cell stain
Chromosome counterstain
• Flow cytometry
• Chromosome banding
• Mycoplasma detection
• Chromosome and nuclei counterstain
• Chromosome banding
• Electrophoresis
• Flow cytometry
• Argon-ion laser excitable
AT-selective
Spectra dependent on secondary structure and sequence
RNA/DNA discrimination
Nuclear stain in tissue
• Permeant
• High Stokes shift
• Long-wavelength spectra
• Flow cytometry
• Impermeant
• Nuclear counterstain
• Impermeant
• Dead-cell stain
• Chromosome and nuclear counterstain
* Excitation (Ex) and emission (Em) maxima, in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous
solution, unless otherwise indicated. † Indication of dyes as “permeant” or “impermeant” are for the most common applications; permeability to cell membranes may vary
considerably with the cell type, dye concentrations and other staining conditions. ‡ Available in aqueous solution for those wishing to avoid potentially hazardous and
mutagenic powders. § After oxidation to ethidium. ** Prior to photolysis; after photolysis the spectra of the dye/DNA complexes are similar to those of ethidium bromide–
DNA complexes.
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EthD-1 binds per four base pairs in dsDNA,6 and the dye’s intercalation is not sequence
selective.69 It was originally reported that only one of the two phenanthridinium rings of
EthD-1 is bound at a time; 6 subsequent reports indicate that bis-intercalation appears to
be involved in staining both double-stranded and triplex nucleic acids.24,68
The spectra and other properties of the EthD-1 and EthD-2 dimers are almost identical
(Figure 8.19). However, the DNA affinity of EthD-2 is about twice that of EthD-1. EthD2 is also about twice as fluorescent bound to dsDNA than to RNA. Because both EthD-1
and EthD-2 can be excited with UV light or by the 488 nm spectral line of the argon-ion
laser, either dye can be used in combination with the TOTO-1, YOYO-1 or SYTOX
Green nucleic acid stains for multicolor experiments (Figure 8.20). The ethidium homodimer dyes are impermeant to cells with intact membranes, a property that makes
EthD-1 useful as a dead-cell indicator in our LIVE/DEAD Viability/Cytotoxicity Kit
(L-3224, Section 15.3, Figure 15.16) and EthD-2 (under our trademark name Dead Red)
a suitable dead-cell indicator in our LIVE/DEAD Reduced Biohazard Cell Viability Kit
#1 (L-7013; Section 15.3; Figure 15.23, Figure 15.24). These dyes have also been used to
detect DNA in solution,69 although they are not as sensitive or as easy to use as our
PicoGreen dsDNA quantitation reagent (Section 8.3).
Ethidium Monoazide: A Photocrosslinking Reagent
Nucleic acids can be covalently photolabeled by various DNA intercalators. Ethidium
monoazide (E-1374, Figure 8.21) is a fluorescent photoaffinity label that, after photolysis, binds covalently to nucleic acids both in solution and in cells that have compromised
membranes.70–74 The quantum yield for covalent photolabeling by ethidium monoazide is
unusually high (>0.4).
The membrane-impermeant ethidium monoazide is reported to only label dead cells
and is therefore particularly useful for assaying the viability of pathogenic cells (Section
15.2). A mixed population of live and dead cells incubated with this reagent can be illuminated with a visible-light source, washed, fixed and then analyzed in order to determine the viability of the cells at the time of photolysis.75 This method not only reduces
some of the hazards inherent in working with pathogenic cells, but also is compatible
with immunocytochemical analyses requiring fixation. We have developed alternative
assays for determining the original viability of fixed samples and provide these in four
LIVE/DEAD Reduced Biohazard Cell Viability Kits (L-7013, L-23101, L-23102 and
L-23105), which are described in Section 15.3.
In addition to its utility as a viability indicator, ethidium monoazide has been used to
irreversibly label the DNA of Candida albicans in order to investigate phagocytic capacity of leukocytes.76 Ethidium monoazide has also been employed to “footprint” drugbinding sites on DNA,77 to probe for ethidium-binding sites in DNA 78 and transfer RNA
(tRNA) 73 and to selectively photo-inactivate the expression of genes in vertebrate cells.79
Acridine Orange: A Dual-Fluorescence Nucleic Acid Stain
Molecular Probes offers highly purified, flow cytometry–grade acridine orange, a
dye that interacts with DNA and RNA by intercalation or electrostatic attractions. In
condensed chromatin, however, the bulk of DNA is packed in a way that does not allow
efficient acridine orange intercalation.80 This cationic dye (Figure 8.22) has green fluorescence with an emission maximum at 525 nm when bound to DNA. Upon association
with RNA, its emission is shifted to ~650 nm (red fluorescence).
Figure 8.21 E-1374 ethidium monoazide bromide.
Figure 8.22 A-1301 acridine orange.
Figure 8.16 H-7593 hexidium iodide.
Figure 8.17 E-1169 ethidium homodimer-1.
Figure 8.18 E-3599 ethidium homodimer-2.
Figure 8.19 Absorption and fluorescence emission
spectra of ethidium homodimer-1 (E-1169) bound
to DNA.
Figure 8.20 Normalized fluorescence emission
spectra of DNA-bound SYTOX Green nucleic acid
stain (S-7020) and ethidium homodimer-1 (EthD1, E-1169). Both spectra were obtained using excitation at 488 nm.
Section 8.1
279
Acridine orange is available as a solid (A-1301) and, for ease of handling, as a
10 mg/mL aqueous solution (A-3568).
Figure 8.23 A-666 acridine homodimer.
Figure 8.24 A-1324 9-amino-6-chloro-2-methoxyacridine (ACMA).
Figure 8.25 H-1398 Hoechst 33258.
AT-Selective Acridine Homodimer
The water-soluble acridine homodimer — bis-(6-chloro-2-methoxy-9-acridinyl)spermine (A-666, Figure 8.23) — is one of several acridine dimers that have been
described in the literature. This dye has extremely high affinity for AT-rich regions of
nucleic acids, making it particularly useful for chromosome banding 81,82 (Section 8.6).
Acridine homodimer emits a blue-green fluorescence when bound to DNA, yielding
fluorescence that is proportional to the fourth power of the AT base-pair content.83 Acridine homodimer has been recommended as an alternative to quinacrine for Q banding
because of its greater brightness and higher photostability.81
AT-Selective ACMA
ACMA (9-amino-6-chloro-2-methoxyacridine, A-1324, Figure 8.24) is a DNA
intercalator that selectively binds to poly(d(A-T)) with a binding affinity constant of
2 × 10 5 M-1 at pH 7.4.84,85 Excitation of the ACMA–DNA complex (excitation/emission
maxima ~419/483 nm) is possible with most UV-light sources, making it compatible for
use with both shorter- and longer-wavelength dyes. ACMA also apparently binds to
membranes in the energized state and becomes quenched if a pH gradient forms.86 It has
been extensively employed to follow cation and anion movement across membranes 86–89
and to study the proton-pumping activity of various membrane-bound ATPases 90,91
(Section 21.3).
Indoles and Imidazoles: Classic Minor Groove–Binding Dyes
DNA-Selective Hoechst Dyes
The bisbenzimide dyes — Hoechst 33258 (Figure 8.25), Hoechst 33342 (Figure 8.26)
and Hoechst 34580 (Figure 8.27) — are cell membrane–permeant, minor groove–binding
DNA stains that fluoresce bright blue upon binding to DNA. Hoechst 33342 has slightly
higher membrane permeability than Hoechst 33258,61 but both dyes are quite soluble in
water (up to 2% solutions can be prepared) and relatively nontoxic. Hoechst 34580 92
(H-21486) has somewhat longer-wavelength spectra than the other Hoechst dyes when
bound to nucleic acids. These Hoechst dyes, which can be excited with the UV spectral
lines of the argon-ion laser and by most conventional fluorescence excitation sources,
exhibit relatively large Stokes shifts (Figure 8.28) (excitation/emission maxima
~350/460 nm), making them suitable for multicolor labeling experiments. The Hoechst
33258 and Hoechst 33342 dyes have complex, pH-dependent spectra when not bound to
nucleic acids, with a much higher fluorescence quantum yield at pH 5 than at pH 8. Their
fluorescence is also enhanced by surfactants such as sodium dodecyl sulfate (SDS).93 These
dyes appear to show a wide spectrum of sequence-dependent DNA affinities and bind with
sufficient strength to poly(d(A-T)) sequences that they can displace several known DNA
intercalators.94 They also exhibit multiple binding modes and distinct fluorescence emission
spectra that are dependent on dye:base pair ratios.95 Hoechst dyes are used in many cellular
applications, including in cell-cycle and apoptosis studies (Section 15.4, Section 15.5) and
they are common nuclear counterstains (Section 8.6). Hoechst 33258, which is selectively
toxic to malaria parasites,96 is also useful for flow-cytometric screening of blood samples
for malaria parasites and for their susceptibility to drugs; 97–99 however, some of our SYTO
dyes are likely to provide superior performance in these assays.
The Hoechst 33258 and Hoechst 33342 dyes are available as solids (H-1398, H-1399),
as FluoroPure grade solids (H-21491, H-21492) and, for ease of handling, as 10 mg/mL
aqueous solutions (H-3569, H-3570).
Figure 8.26 H-1399 Hoechst 33342.
Figure 8.27 H-21486 Hoechst 34580.
Figure 8.28 Absorption and fluorescence emission
spectra of Hoechst 33258 (H-1398, H-3569, H-21491)
bound to DNA.
280
AT-Selective DAPI
DAPI (4′,6-diamidino-2-phenylindole; D-1306, D-3571; FluoroPure grade, D-21490;
Figure 8.29) shows blue fluorescence (Figure 14.1) upon binding DNA and can be excited with a mercury-arc lamp or with the UV lines of the argon-ion laser. Like the Hoechst
dyes, the blue-fluorescent DAPI stain apparently associates with the minor groove of
dsDNA (Figure 8.30), preferentially binding to AT clusters; 100 there is evidence that
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DAPI also binds to DNA sequences that contain as few as two consecutive AT base pairs,
perhaps employing a different binding mode.101–103 DAPI is thought to employ an intercalating binding mode with RNA that is AU selective.104
The selectivity of DAPI for DNA over RNA is reported to be greater than that displayed
by ethidium bromide and propidium iodide.105 Furthermore, the DAPI–RNA complex
exhibits a longer-wavelength fluorescence emission maximum than the DAPI–dsDNA
complex (~500 nm versus ~460 nm) and a quantum yield that is only about 20% as high.106
Binding of DAPI to dsDNA produces an ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor
groove.107 Although the Hoechst dyes may be somewhat brighter in some applications,
their photostability when bound to dsDNA is less than that of DAPI. In the presence of
appropriate salt concentrations, DAPI does not usually exhibit fluorescence enhancement
upon binding to ssDNA or GC base pairs.108 However, the fluorescence of DAPI does
increase significantly upon binding to detergents,109 dextran sulfate,110 polyphosphates
and other polyanions.111 A review by Kapuscinski discusses the mechanisms of DAPI
binding to nucleic acids, its spectral properties and its uses in flow cytometry and for
chromosome staining.112 DAPI is an excellent nuclear counterstain and shows a distinct
banding pattern in chromosomes (Section 8.6, Figure 8.127). DAPI is quite soluble in
water but has limited solubility in phosphate-buffered saline.
We also offer DAPI pre-mixed with our SlowFade and SlowFade Light antifade reagents (S-24635, S-24636). This permits simultaneous staining and protection of the
stained sample from photobleaching.
Figure 8.29 D-1306 4′,6-diamidino-2-phenylindole
(DAPI).
Other Nucleic Acid Stains
The Intercalators 7-Aminoactinomycin D and Actinomycin D
7-AAD (7-aminoactinomycin D, A-1310; Figure 8.31) is a fluorescent intercalator
that undergoes a spectral shift upon association with DNA. 7-AAD–DNA complexes can
be excited by the argon-ion laser and emit beyond 610 nm (Figure 8.15, Figure 8.32;
Table 8.4), making this nucleic acid stain useful for multicolor fluorescence microscopy
(Figure 8.33), confocal laser-scanning microscopy and immunophenotyping by flow
cytometry.113–118 7-AAD appears to be generally excluded from live cells, although it has
been reported to label the nuclear region of live cultured mouse L cells and salivary gland
polytene chromosomes of Chironomus thummi larvae.119 7-AAD binds selectively to GC
regions of DNA,120 yielding a distinct banding pattern in polytene chromosomes and
chromatin.119,121 This sequence selectivity has been exploited for chromosome banding
studies 122 (Section 8.6).
Actinomycin D (A-7592) is a nonfluorescent intercalator that exhibits high GC selectivity and causes distortion at its binding site.123 Binding of the nonfluorescent actinomycin D to nucleic acids changes the absorbance of the dye.124 Like 7-AAD, actinomycin D
has been used for chromosome banding studies.125 Binding of actinomycin D to ssDNA
is reported to inhibit reverse transcriptase and other polymerases.126
Figure 8.30 X-ray crystal structure of DAPI
(D-1306, D-3571, D-21490) bound to DNA. X-ray
crystallography shows that DAPI binds to DNA in
the minor groove. The image was derived from data
submitted to the Protein Data Bank (www.rcsb.org/
pdb/, (Nucleic Acids Res 28, 235 (2000))), number
PDB 1D30. The initial structure was described in J
Biomol Struct Dyn 7, 477 (1989).
Figure 8.31 A-1310 7-aminoactinomycin D (7-AAD).
Figure 8.32 Absorption and fluorescence
emission spectra of 7-aminoactinomycin D
(7-AAD, A-1310) bound to DNA.
7-Aminoactinomycin D is frequently
used to detect apoptotic cells
(Section 15.5).
Section 8.1
281
Figure 8.34 H-22845 hydroxystilbamidine, methanesulfonate.
Figure 8.35 Fluorescence spectra of hydroxystilbamidine bound to different forms of DNA.
Hydroxystilbamidine (H-22845) was incubated
with either calf thymus DNA (red) or a hybrid of
poly(d(A)) and poly(d(T)) homopolymers (blue) in
50 mM sodium acetate, pH 5.0. The fluorescence
emission spectra changes when the dye is bound
to AT-rich DNA versus calf-thymus genomic DNA.
Figure 8.36 L-7595 LDS 751.
Multicolor Hydroxystilbamidine
Hydroxystilbamidine 127 (H-22845, Figure 8.34) — a trypanocidal drug that has previously been sold for research use as a neuronal tracer 128,129 under the trademark FluoroGold (a trademark of FluoroChrome, Inc.) — is an interesting probe of nucleic acid
conformation with nucleic acid staining properties that were first described in 1973.130
Hydroxystilbamidine, a nonintercalating dye, exhibits AT-selective binding that is reported to favor regions of nucleic acids that have secondary structure. The interaction between hydroxystilbamidine and DNA has been investigated using binding isotherms 131
and temperature-jump relaxation studies.132
Hydroxystilbamidine has some unique spectral properties upon binding nucleic acids.
At pH 5, the free dye exhibits UV excitation maxima at ~330 nm and ~390 nm, with dual
emission at ~450 nm and ~600 nm (Figure 8.35). Although the red-fluorescent component remains present when bound to DNA, it is never observed when the dye is bound to
RNA, allowing potential discrimination to be made between these two types of nucleic
acids. The enhancement of its metachromatic fluorescence upon binding to DNA is
proportional to the square of the AT base-pair content. Hydroxystilbamidine is reported
to exhibit red fluorescence when bound to calf thymus DNA and T5 DNA, orange fluorescence with M. lysodekticus DNA and blue-violet fluorescence on poly(d(A-T)).130 It
has been used for the treatment of myeloma, binding selectively to myeloma cells in the
bone marrow.133
Because hydroxystilbamidine has been unavailable commercially, or its identity has
been obscured by a trademark, its use as a nucleic acid stain in cellular applications has
not been extensively tested. However, Murgatroyd described use of its metachromatic
fluorescence properties for the selective permanent staining of DNA (with yellow fluorescence), mucosubstances and elastic fibers in paraffin sections.134 He also reported that
hydroxystilbamidine (as its isethionate salt, which is not available from Molecular
Probes) is nonmutagenic in Salmonella typhimurium by the Ames test.134
Long-Wavelength LDS 751
LDS 751 (L-7595, Figure 8.36) is a cell-permeant nucleic acid stain that has been
used to discriminate intact nucleated cells from nonnucleated and damaged nucleated
cells,135,136 as well as to identify distinct cell types in mixed populations of neutrophils,
leukocytes and monocytes by flow cytometry.137 LDS 751, which has its peak excitation at
~543 nm on dsDNA, can be excited by the argon-ion laser at 488 nm and is particularly
useful in multicolor analyses due to its long-wavelength emission maximum (~712 nm).
Binding of LDS 751 to dsDNA results in an ~20-fold fluorescence enhancement. When
LDS 751 binds to RNA, we have observed a significant red shift in its excitation maxi-
Figure 8.33 Panel of confocal micrographs showing cells from wheat
root tips in seven stages of the cell cycle. DNA was stained with 7-aminoactinomycin D (A-1310), and microtubules were labeled with an anti–
β-tubulin antibody in conjunction with a fluorescein-labeled secondary
antibody. Cells vary in width from about 15 µm to about 25 µm. The
stages are (from top left): interphase cortical microtubule array; preprophase band of microtubules (predicts future plane of division);
metaphase mitotic spindle; telophase, showing early phragmoplast and
cell plate; fully developed phragmoplast during cytokinesis; late cytokinesis (plane of division matching plane of earlier pre-prophase band); restoration of cortical arrays in daughter cells. Image contributed by B.E.S.
Gunning, Plant Cell Biology Group, Research School of Biological Sciences, Australian National University. Used with permission from Gunning, B.E.S. and Steer, M.W., Plant Cell Biology — Structure and Function, Jones and Bartlett Publishers (1995).
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mum to 590 nm and blue shift in its emission maxima to 607 nm, which may permit its
use to discriminate DNA and RNA in cells. A report has ascribed the name LDS 751 to
another dye called styryl 8; 138 however, their chemical structures are not the same.
NeuroTrace Fluorescent Nissl Stains
The Nissl substance, described by Franz Nissl over one hundred years ago, is unique
to neuronal cells. Composed of an extraordinary amount of rough endoplasmic reticulum,
the Nissl substance reflects the unusually high protein synthesis capacity of neuronal cells.
Various fluorescent or chromophoric “Nissl stains” have been used to stain the Nissl
substance in tissue preparations, thereby identifying neuronal cells. Because the Nissl
substance redistributes within the cell body in injured or regenerating neurons, Nissl stains
can also be used to probe the physiological state of the neuron. Stains used for this purpose include acridine orange,139 ethidium bromide,139 neutral red (N-3246, Section 15.2),
cresyl violet,140 methylene blue, safranin-O and toluidine blue-O.141 We have developed
five fluorescent Nissl stains that not only provide a wide spectrum of fluorescent colors for
staining neurons, but also are more sensitive than the conventional dyes:
• NeuroTrace 435/455 blue fluorescent Nissl stain (N-21479, Figure 8.112)
• NeuroTrace 500/525 green fluorescent Nissl stain (N-21480; Figure 7.82, Figure 8.37,
Figure 14.41, Figure 14.56)
• NeuroTrace 515/535 yellow fluorescent Nissl stain (N-21481, Figure 14.42)
• NeuroTrace 530/615 red fluorescent Nissl stain (N-21482; Figure 14.1, Figure 14.38)
• NeuroTrace 640/660 deep red-fluorescent Nissl stain (N-21483)
Although staining by the Nissl stains is completely eliminated by pretreatment of
tissue specimens with RNase, these dyes are not specific stains for RNA in solution.
Figure 8.37 Pyramidal cells of the hippocampus
and dentate gyrus in a transverse cryosection of
paraformaldehyde-fixed mouse brain. NeuroTrace
green fluorescent Nissl stain (N-21480) is localized
to neuronal somata, while non-neuronal cells can
be identified by the presence of DAPI-stained nuclei. This image is a composite of images taken using a 10× objective and filters appropriate for fluorescein and DAPI.
References
1. Mutat Res 439, 37 (1999); 2. US 5,410,030 and
CA 2,119,126; 3. Nucleic Acids Res 23, 1215
(1995); 4. Nucleic Acids Res 19, 327 (1991);
5. Biochemistry 17, 5071 (1978); 6. Biochemistry
17, 5078 (1978); 7. Nucleic Acids Res 20, 2803
(1992); 8. Proc Natl Acad Sci U S A 87, 3851
(1990); 9. Nucleic Acids Res 21, 5720 (1993);
10. Nature 359, 859 (1992); 11. Biotechniques 10,
616 (1991); 12. J Phys Chem 99, 17936 (1995);
13. Cytometry 7, 508 (1986); 14. Appl Environ
Microbiol 66, 2283 (2000); 15. Appl Environ
Microbiol 61, 3623 (1995); 16. Anal Biochem
221, 78 (1994); 17. EP Application No. EP 0 634
640 A1. 18. J Am Chem Soc 116, 8459 (1994);
19. J Phys Chem 98, 10313 (1994); 20. J Biomol
Struct Dyn 16, 205 (1998); 21. J Biomol Struct
Dyn 16, 205 (1998); 22. Anal Chem 67, 663A
(1995); 23. FASEB J 7, A1087, abstract #205
(1993); 24. Nucleic Acids Res 23, 2413 (1995);
25. Biochemistry 34, 8542 (1995); 26. Biochemistry 37, 16863 (1998); 27. Cytometry 37, 230
(1999); 28. Bioconjug Chem 10, 824 (1999);
29. Nucleic Acids Res 24, 859 (1996); 30. Nucleic Acids Res 23, 753 (1995); 31. Acta Chem
Scand 52, 641 (1998); 32. Biochem Mol Biol Int
34, 1189 (1994); 33. Biopolymers 41, 481 (1997);
34. US 5,321,130; 35. Appl Environ Microbiol
60, 3284 (1994); 36. Cancer Res 57, 3804 (1997);
37. Blood 87, 4959 (1996); 38. J Immunol Methods 185, 249 (1995); 39. J Exp Med 182, 1759
(1995); 40. Br J Pharmacol 130, 513 (2000);
41. Br J Pharmacol 125, 1194 (1998); 42. J Biol
Chem 276, 125 (2001); 43. Cytometry 17, 185
(1994); 44. Cytometry 17, 310 (1994); 45. Cy-
tometry 15, 267 (1994); 46. Handbook of Fluorescent Probes and Research Chemicals, 5th Ed.,
1992-1994, Haugland RP (Complete Volume),
(1992); 47. J Phys Chem B 104, 7221 (2000);
48. Limnol Oceanogr 40, 1050 (1995); 49. Anal
Biochem 293, 8 (2001); 50. Anal Biochem 286,
138 (2000); 51. US 5,436,134 and US 5,445,946;
52. Proc Natl Acad Sci U S A 94, 14804 (1997);
53. J Neurosci 16, 7812 (1996); 54. Cytometry
21, 265 (1995); 55. Neuron 15, 961 (1995);
56. Cytometry 20, 218 (1995); 57. Anal Biochem
287, 179 (2000); 58. Anal Biochem 281, 26
(2000); 59. J Mol Biol 13, 269 (1965); 60. Biochemistry 29, 981 (1990); 61. Methods Cell Biol
30, 417 (1989); 62. Chromatographia 29, 167
(1990); 63. Anal Biochem 162, 453 (1987);
64. US 5,437,980; 65. J Immunol Methods 170,
117 (1994); 66. FEMS Microbiol Lett 101, 173
(1992); 67. J Histochem Cytochem 34, 1109
(1986); 68. Bioorg Med Chem 3, 701 (1995);
69. Anal Biochem 94, 259 (1979); 70. Biochemistry 30, 5644 (1991); 71. Photochem Photobiol 43,
7 (1986); 72. J Biol Chem 259, 11090 (1984);
73. Photochem Photobiol 36, 31 (1982); 74. J Mol
Biol 92, 319 (1975); 75. Cytometry 12, 133
(1991); 76. Cytometry 11, 610 (1990); 77. Eur J
Biochem 182, 437 (1989); 78. Biochemistry 19,
3221 (1980); 79. Proc Natl Acad Sci U S A 97,
9504 (2000); 80. Exp Cell Res 194, 147 (1991);
81. Methods Mol Biol 29, 83 (1994); 82. Biochemistry 18, 3354 (1979); 83. Proc Natl Acad
Sci U S A 72, 2915 (1975); 84. Eur J Biochem
180, 359 (1989); 85. J Biomol Struct Dyn 5, 361
(1987); 86. Biochim Biophys Acta 722, 107
(1983); 87. Biochim Biophys Acta 1143, 215
(1993); 88. Eur Biophys J 19, 189 (1991); 89. Biochemistry 19, 1922 (1980); 90. J Biol Chem 269,
10221 (1994); 91. Biochim Biophys Acta 1183,
161 (1993); 92. Cytometry 44, 133 (2001);
93. Photochem Photobiol 73, 339 (2001); 94. Biochemistry 29, 9029 (1990); 95. J Histochem
Cytochem 33, 333 (1985); 96. Mol Biochem
Parasitol 58, 7 (1993); 97. Am J Trop Med Hyg
43, 602 (1990); 98. Cytometry 14, 276 (1993);
99. Methods Cell Biol 42 Pt B, 295 (1994);
100. Biochemistry 26, 4545 (1987); 101. Biochemistry 32, 2987 (1993); 102. J Biol Chem 268,
3944 (1993); 103. Biochemistry 29, 8452 (1990);
104. Biochemistry 31, 3103 (1992); 105. Nucleic
Acids Res 6, 3535 (1979); 106. J Histochem
Cytochem 38, 1323 (1990); 107. Biochem Biophys Res Commun 170, 270 (1990); 108. Nucleic
Acids Res 6, 3519 (1979); 109. Nucleic Acids
Res 5, 3775 (1978); 110. Can J Microbiol 26, 912
(1980); 111. Biochim Biophys Acta 721, 394
(1982); 112. Biotech Histochem 70, 220 (1995);
113. Exp Parasitol 97, 141 (2001); 114. Br J
Haematol 104, 530 (1999); 115. Cytometry 12,
221 (1991); 116. Cytometry 12, 172 (1991);
117. J Immunol 136, 2769 (1986); 118. J Histochem Cytochem 23, 793 (1975); 119. Histochem J 17, 131 (1985); 120. Biopolymers 18,
1749 (1979); 121. Cytometry 20, 296 (1995);
122. Chromosoma 68, 287 (1978); 123. J Mol
Biol 225, 445 (1992); 124. Biochemistry 32, 5554
(1993); 125. Cancer Genet Cytogenet 1, 187
(1980); 126. Biochemistry 35, 3525 (1996);
127. We have determined that the product sold
Section 8.1
283
References — continued
prior to July 2001 as hydroxystilbamidine (under
catalog number H-7599) was in fact aminostilbamidine. To rectify this situation and avoid
confusion in the future, we have prepared authentic hydroxystilbamidine. Aminostilbamidine is
offered under a new catalog number. Product
number H-7599 has been discontinued. This
cationic dye is also frequently used as a retro-
grade neuronal tracer. We recommend that customers who have previously used our product
number H-7599 for retrograde neuronal tracing
applications should in the future use aminostilbamidine. 128. J Neurocytol 18, 333 (1989);
129. US 4,716,905; 130. Biochemistry 12, 4827
(1973); 131. Biochim Biophys Acta 407, 24
(1975); 132. Biochim Biophys Acta 407, 43
(1975); 133. J Lab Clin Med 37, 562 (1951);
134. Histochemistry 74, 107 (1982); 135. J
Immunol Methods 123, 103 (1989); 136. Cytometry 9, 477 (1988); 137. J Immunol Methods 163,
155 (1993); 138. J Photochem Photobiol A 84, 45
(1994); 139. Proc Natl Acad Sci U S A 77, 2260
(1980); 140. J Neurosci Methods 33, 129 (1990);
141. J Neurosci Methods 72, 49 (1997).
Data Table — 8.1 Nucleic Acid Stains
Cat #
A-666
A-1301
A-1310
A-1324
A-3568
A-7592
B-3582
B-3583
B-3586
B-3587
D-1168
D-1306
D-3571
D-11347
D-21490
D-23107
E-1169
E-1305
E-1374
E-3565
E-3599
H-1398
H-1399
H-3569
H-3570
H-7593
H-21486
H-21491
H-21492
H-22845
J-11372
J-11373
L-7595
L-11376
L-11377
N-21479
N-21480
N-21481
N-21482
N-21483
N-21485
O-7582
P-1304
P-3566
P-3580
P-3581
P-3584
P-3585
P-7581
P-11495
P-21493
R-11491
S-7020
S-7555
S-7556
S-7557
284
MW
685.69
301.82
1270.45
258.71
301.82
1255.43
1202.66
595.32
1254.73
621.36
315.42
350.25
457.49
315.42
350.25
315.42
856.77
394.31
420.31
394.31
1292.71
623.96
615.99
623.96
615.99
497.42
560.96
623.96
615.99
472.53
1272.63
630.31
471.98
1462.54
725.27
see Notes
see Notes
see Notes
see Notes
see Notes
651.01
see Notes
668.40
668.40
1170.53
579.26
1222.61
605.30
see Notes
see Notes
668.40
see Notes
~600
~450
~500
~350
Storage
L
L
F,L
L
RR,L
F,L
F,D,L
F,D,L
F,D,L
F,D,L
FF,L,AA
L
L
FF,L,AA
L
FF,D,L,AA
F,D,L
L
F,LL
RR,L
F,D,L
L
L
RR,L
RR,L
L
L
L
L
F,D,L
F,D,L
F,D,L
L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
L
F,D,L
L
RR,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
Soluble
DMSO, DMF
H2O, EtOH
DMF, DMSO
DMF, DMSO
H2O
DMF, DMSO
DMSO
DMSO
DMSO
DMSO
DMF, DMSO
H2O, DMF
H2O, MeOH
DMF, DMSO
H2O, DMF
DMSO
DMSO
H2O, DMSO
DMF, EtOH
H2O
DMSO
H2O, DMF
H2O, DMF
H2O
H2O
DMSO
DMSO
H2O, DMF
H2O, DMF
H2O, DMSO
DMSO
DMSO
DMSO, EtOH
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
H2O, DMSO
H2O
DMSO
DMSO
DMF
DMSO
DMSO
DMSO
H2O, DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Abs
431
500
546
412
500
442
462
462
570
575
355
358
358
355
358
355
528
518
462
518
535
352
350
352
350
518
392
352
350
360
530
532
543
566
568
435
497
515
530
644
355
498
535
535
434
435
534
539
502
502
535
500
504
512
494
515
EC
ND
53,000
25,000
8,200
53,000
23,000
114,000
58,000
148,000
81,000
14,000
21,000
20,000
14,000
21,000
14,000
7,000
5,200
5,400
5,200
8,000
40,000
45,000
40,000
45,000
3,900
47,000
40,000
45,000
27,000
171,000
94,000
46,000
108,000
103,000
see Notes
see Notes
see Notes
see Notes
see Notes
36,000
see Notes
5,400
5,400
92,000
50,000
146,000
88,000
see Notes
see Notes
5,400
see Notes
67,000
64,000
43,000
43,000
Em
498
526
647
471
526
none
481
481
604
599
see Notes
461
461
see Notes
461
see Notes
617
605
625
605
624
461
461
461
461
600
440
461
461
625
545
544
712
580
581
457
524
535
619
663
495
518
617
617
456
455
570
567
523
523
617
525
523
530
517
535
Solvent
H2O/DNA
H2O/DNA
H2O/DNA
MeOH
H2O/DNA
MeOH
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
MeCN
H2O/DNA
H2O/DNA
MeCN
H2O/DNA
MeCN
H2O/DNA
H2O/DNA
pH 7
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/RNA
H2O/RNA
H2O/RNA
H2O/RNA
H2O/RNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/RNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
Chapter 8 — Nucleic Acid Detection and Genomics Technology
Notes
1, 2
3, 4
3
5
3, 4, 6
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
9, 10
3, 11
3, 11
9, 10
3, 11, 12
10, 13
3, 7, 14
3, 15
16
3, 6, 15
3, 6, 7, 14
3, 17
3, 17
3, 6, 17
3, 6, 17
3, 18
3
3, 12, 17
3, 12, 17
3, 19
3, 6, 7, 8
3, 6, 7, 8
3
3, 6, 7, 8
3, 6, 7, 8
6, 8, 20
6, 8, 20
6, 8, 20
6, 8, 20
6, 8, 20
3
6, 8, 20
3, 21
3, 6, 21
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
6, 8, 20
6, 8, 20
3, 12, 21
6, 8, 20
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
www.probes.com
Cat #
S-7558
S-7559
S-7560
S-7573
S-7574
S-7575
S-7576
S-7577
S-7578
S-7579
S-11341
S-11342
S-11343
S-11344
S-11345
S-11346
S-11348
S-11351
S-11352
S-11353
S-11354
S-11355
S-11356
S-11361
S-11362
S-11363
S-11364
S-11365
S-11366
S-11368
S-21500
S-21501
S-21502
T-3600
T-3602
T-3604
T-3605
T-7596
Y-3601
Y-3603
Y-3606
Y-3607
MW
~400
~550
~450
~400
~300
~400
~500
~400
~450
~650
~550
~500
~500
~550
~550
~400
~400
~250
~450
~350
~400
~300
~300
~400
~300
~350
~350
~500
~350
~500
~600
~550
~600
1302.78
645.38
1354.85
671.42
697.46
1270.65
629.32
1322.73
655.36
Storage
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
Soluble
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Abs
499
490
521
508
500
488
517
516
488
621
622
652
620
649
654
598
445
419
426
430
437
445
452
531
530
541
543
567
567
547
494
484
486
514
515
642
642
747
491
491
612
612
EC
46,000
58,000
57,000
75,000
54,000
74,000
60,000
55,000
42,000
88,000
112,000
83,000
85,000
76,000
119,000
84,000
38,000
33,000
34,000
31,000
48,000
56,000
43,000
89,000
82,000
76,000
68,000
95,000
86,000
79,000
57,000
39,000
56,000
117,000
63,000
154,000
102,000
108,000
99,000
52,000
167,000
100,000
Em
520
515
556
527
522
509
549
546
518
634
645
678
647
680
675
620
470
445
455
460
464
472
484
545
544
560
559
582
583
570
519
520
526
533
531
660
661
770
509
509
631
631
Solvent
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
H2O/DNA
Notes
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 6, 8, 22
3, 8, 22
3, 8, 22
3, 8, 22
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
3, 6, 7, 8
For definitions of the contents of this data table, see “How to Use This Book” on page viii.
Notes
1. ND = not determined.
2. A-666 in MeOH: Abs = 418 nm (EC = 12,000 cm-1M-1), Em = 500 nm.
3. Spectra represent aqueous solutions of nucleic acid–bound dye. EC values are derived by comparing the absorbance of the nucleic acid–bound dye with that of free dye in a
reference solvent (H2O or MeOH).
4. Acridine orange bound to RNA has Abs ~460 nm, Em ~650 nm (Methods Cell Biol 41, 401 (1994); Cytometry 2, 201 (1982)).
5. Spectra of this compound are in methanol acidified with a trace of HCl.
6. This product is supplied as a ready-made solution in the solvent indicated under Soluble.
7. Although this compound is soluble in water, preparation of stock solutions in water is not recommended because of possible adsorption onto glass or plastic.
8. This product is essentially nonfluorescent except when bound to DNA or RNA.
9. This compound is susceptible to oxidation, especially in solution. Store solutions under argon or nitrogen. Oxidation appears to be catalyzed by illumination.
10. Dihydroethidium has blue fluorescence (Em ~420 nm) until oxidized to ethidium (E-1305). The reduced dye does not bind to nucleic acids (FEBS Lett 26, 169 (1972)).
11. DAPI in H2O: Abs = 344 nm (EC = 23,000 cm-1M-1), Em = 450 nm. QY increases ~20-fold on binding to dsDNA (Ital J Biochem 31, 90 (1982)).
12. This product is specified to equal or exceed 98% analytical purity by HPLC.
13. This product is supplied as a ready-made solution in DMSO with sodium borohydride added to inhibit oxidation.
14. E-1169 in H2O: Abs = 493 nm (EC = 9100 cm-1M-1). E-3599 in H2O: Abs = 498 nm (EC = 10,800 cm-1M-1). Both compounds are very weakly fluorescent in H2O. QY increases
>40-fold on binding to dsDNA.
15. Ethidium bromide in H2O: Abs = 480 nm (EC = 5600 cm-1M-1), Em = 620 nm (weakly fluorescent). Fluorescence is enhanced >10-fold on binding to dsDNA.
16. E-1374 spectral data are for the free dye. Fluorescence is weak, but intensity increases ~15-fold on binding to DNA. After photocrosslinking to DNA, Abs = 504 nm (EC ~4000 cm-1M-1),
Em = 600 nm (Nucleic Acids Res 5, 4891 (1978); Biochemistry 19, 3221 (1980)).
17. MW is for the hydrated form of this product.
18. H-7593 in H2O: Abs = 482 nm (EC = 5500 cm-1M-1), Em = 625 nm (weakly fluorescent).
19. Nucleic acid–bound hydroxystilbamidine has a second fluorescence emission peak at ~450 nm. The relative amplitudes of the two emission peaks are dependent on the nucleotide
content of the nucleic acid (Biochemistry 12, 4827 (1973)).
20. The active ingredient of this product is an organic dye with MW <1000. The exact MW value and extinction coefficient of this dye is proprietary.
21. Propidium iodide in H2O: Abs = 493 nm (EC = 5900 cm-1M-1), Em = 636 nm (weakly fluorescent). Fluorescence is enhanced >10-fold on binding to dsDNA.
22. MW: The preceding ~ symbol indicates an approximate value, not including counterions.
Section 8.1
285
Product List — 8.1 Nucleic Acid Stains
Product information for SYTO dye products is given in Table 8.3.
Cat #
Product Name
A-666
A-1301
A-3568
A-7592
A-1310
A-1324
B-3582
B-3586
B-3583
B-3587
D-1306
D-21490
D-3571
D-1168
D-11347
D-23107
E-1305
E-3565
E-1169
E-3599
E-1374
H-7593
H-3569
H-1398
H-21491
H-3570
H-1399
H-21492
H-21486
H-22845
J-11372
J-11373
L-7595
L-11376
L-11377
N-21479
N-21480
N-21481
N-21482
N-21483
N-21485
N-7565
O-11492
O-7582
P-7589
P-11496
P-7581
P-11495
P-3580
P-3584
P-3581
P-3585
P-1304
P-21493
P-3566
R-21495
R-21496
R-11490
R-11491
S-24635
S-24636
S-7550
S-11494
acridine homodimer (bis-(6-chloro-2-methoxy-9-acridinyl)spermine) .................................................................................................................
10 mg
acridine orange .....................................................................................................................................................................................................
1g
acridine orange *10 mg/mL solution in water* ....................................................................................................................................................
10 mL
actinomycin D ......................................................................................................................................................................................................
10 mg
7-aminoactinomycin D (7-AAD) ...........................................................................................................................................................................
1 mg
9-amino-6-chloro-2-methoxyacridine (ACMA) .....................................................................................................................................................
100 mg
BOBO™-1 iodide (462/481) *1 mM solution in DMSO* .......................................................................................................................................
200 µL
BOBO™-3 iodide (570/602) *1 mM solution in DMSO* .......................................................................................................................................
200 µL
BO-PRO™-1 iodide (462/481) *1 mM solution in DMSO* ...................................................................................................................................
1 mL
BO-PRO™-3 iodide (575/599) *1 mM solution in DMSO* ...................................................................................................................................
1 mL
4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) .......................................................................................................................................
10 mg
4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) *FluoroPure™ grade* ....................................................................................................
10 mg
4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate) ....................................................................................................................................
10 mg
dihydroethidium (hydroethidine) ..........................................................................................................................................................................
25 mg
dihydroethidium (hydroethidine) *special packaging* ......................................................................................................................................... 10 x 1 mg
dihydroethidium (hydroethidine) *5 mM stabilized solution in DMSO* ...............................................................................................................
1 mL
ethidium bromide .................................................................................................................................................................................................
1g
ethidium bromide *10 mg/mL solution in water* .................................................................................................................................................
10 mL
ethidium homodimer-1 (EthD-1) ..........................................................................................................................................................................
1 mg
ethidium homodimer-2 (EthD-2) *1 mM solution in DMSO* ...............................................................................................................................
200 µL
ethidium monoazide bromide (EMA) ....................................................................................................................................................................
5 mg
hexidium iodide ....................................................................................................................................................................................................
5 mg
Hoechst 33258 (bis-benzimide) *10 mg/mL solution in water* ...........................................................................................................................
10 mL
Hoechst 33258, pentahydrate (bis-benzimide) .....................................................................................................................................................
100 mg
Hoechst 33258, pentahydrate (bis-benzimide) *FluoroPure™ grade* ..................................................................................................................
100 mg
Hoechst 33342 *10 mg/mL solution in water* .....................................................................................................................................................
10 mL
Hoechst 33342, trihydrochloride, trihydrate .........................................................................................................................................................
100 mg
Hoechst 33342, trihydrochloride, trihydrate *FluoroPure™ grade* ......................................................................................................................
100 mg
Hoechst 34580 .....................................................................................................................................................................................................
5 mg
hydroxystilbamidine, methanesulfonate ...............................................................................................................................................................
10 mg
JOJO™-1 iodide (529/545) *1 mM solution in DMSO* ........................................................................................................................................
200 µL
JO-PRO™-1 iodide (530/546) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
LDS 751 ...............................................................................................................................................................................................................
10 mg
LOLO™-1 iodide (565/579) *1 mM solution in DMSO* .......................................................................................................................................
200 µL
LO-PRO™-1 iodide (567/580) *1 mM solution in DMSO* ...................................................................................................................................
1 mL
NeuroTrace™ 435/455 blue fluorescent Nissl stain *solution in DMSO* .............................................................................................................
1 mL
NeuroTrace™ 500/525 green fluorescent Nissl stain *solution in DMSO* ...........................................................................................................
1 mL
NeuroTrace™ 515/535 yellow fluorescent Nissl stain *solution in DMSO* ..........................................................................................................
1 mL
NeuroTrace™ 530/615 red fluorescent Nissl stain *solution in DMSO* ...............................................................................................................
1 mL
NeuroTrace™ 640/660 deep-red fluorescent Nissl stain *solution in DMSO* ......................................................................................................
1 mL
nuclear yellow (Hoechst S769121, trihydrochloride, trihydrate) ...........................................................................................................................
10 mg
Nucleic Acid Stains Dimer Sampler Kit .................................................................................................................................................................
1 kit
OliGreen® ssDNA Quantitation Kit *200–2000 assays* ........................................................................................................................................
1 kit
1 mL
OliGreen® ssDNA quantitation reagent *200–2000 assays* .................................................................................................................................
PicoGreen® dsDNA Quantitation Kit *200–2000 assays* .....................................................................................................................................
1 kit
PicoGreen® dsDNA Quantitation Kit *200–2000 assays* *special packaging* ....................................................................................................
1 kit
1 mL
PicoGreen® dsDNA quantitation reagent *200–2000 assays* ..............................................................................................................................
PicoGreen® dsDNA quantitation reagent *200–2000 assays* *special packaging* ............................................................................................. 10 x 100 µL
POPO™-1 iodide (434/456) *1 mM solution in DMSO* .......................................................................................................................................
200 µL
POPO™-3 iodide (534/570) *1 mM solution in DMF* ..........................................................................................................................................
200 µL
PO-PRO™-1 iodide (435/455) *1 mM solution in DMSO* ...................................................................................................................................
1 mL
PO-PRO™-3 iodide (539/567) *1 mM solution in DMSO* ...................................................................................................................................
1 mL
propidium iodide ..................................................................................................................................................................................................
100 mg
propidium iodide *FluoroPure™ grade* ...............................................................................................................................................................
100 mg
propidium iodide *1.0 mg/mL solution in water* .................................................................................................................................................
10 mL
RediPlate™ 96 PicoGreen® dsDNA Assay Kit *one 96-well microplate* ..............................................................................................................
1 kit
RediPlate™ 384 PicoGreen® dsDNA Assay Kit *one 384-well microplate* ..........................................................................................................
1 kit
RiboGreen® RNA Quantitation Kit *200–2000 assays* ........................................................................................................................................
1 kit
RiboGreen® RNA quantitation reagent *200–20,000 assays* ..............................................................................................................................
1 mL
SlowFade® Antifade Kit with DAPI ........................................................................................................................................................................
1 kit
SlowFade® Light Antifade Kit with DAPI ...............................................................................................................................................................
1 kit
SYBR® DX DNA blot stain *1000X concentrate in DMSO* ...................................................................................................................................
1 mL
SYBR® Gold nucleic acid gel stain *10,000X concentrate in DMSO* ...................................................................................................................
500 µL
286
Unit Size
Chapter 8 — Nucleic Acid Detection and Genomics Technology
www.probes.com
Cat #
Product Name
S-7563
S-7567
S-7585
S-7564
S-7568
S-7586
S-7580
S-21500
S-21501
S-21502
S-11340
S-11348
S-7020
S-11368
T-3602
T-3605
T-7596
T-3600
T-3604
Y-3603
Y-3607
Y-3601
Y-3606
SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* ...............................................................................................................
500 µL
SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* ...............................................................................................................
1 mL
SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* *special packaging* .............................................................................. 20 x 50 µL
SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* .........................................................................................................................
500 µL
SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* .........................................................................................................................
1 mL
SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* *special packaging* ........................................................................................ 20 x 50 µL
SYBR® Green Nucleic Acid Gel Stain Starter Kit ...................................................................................................................................................
1 kit
SYBR® 101, succinimidyl ester ............................................................................................................................................................................
1 mg
SYBR® 102, succinimidyl ester ............................................................................................................................................................................
1 mg
SYBR® 103, succinimidyl ester ............................................................................................................................................................................
1 mg
SYTO® Red Fluorescent Nucleic Acid Stain Sampler Kit *SYTO® dyes 17 and 59–64* *50 µL each* .................................................................
1 kit
SYTOX® Blue nucleic acid stain *5 mM solution in DMSO* .................................................................................................................................
250 µL
SYTOX® Green nucleic acid stain *5 mM solution in DMSO* ..............................................................................................................................
250 µL
SYTOX® Orange nucleic acid stain *5 mM solution in DMSO* ............................................................................................................................
250 µL
TO-PRO®-1 iodide (515/531) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
TO-PRO®-3 iodide (642/661) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
TO-PRO®-5 iodide (745/770) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
TOTO®-1 iodide (514/533) *1 mM solution in DMSO* ........................................................................................................................................
200 µL
TOTO®-3 iodide (642/660) *1 mM solution in DMSO* ........................................................................................................................................
200 µL
YO-PRO®-1 iodide (491/509) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
YO-PRO®-3 iodide (612/631) *1 mM solution in DMSO* ....................................................................................................................................
1 mL
YOYO®-1 iodide (491/509) *1 mM solution in DMSO* ........................................................................................................................................
200 µL
YOYO®-3 iodide (612/631) *1 mM solution in DMSO* ........................................................................................................................................
200 µL
8.2
Labeling Oligonucleotides and
Nucleic Acids
To facilitate the preparation of optimally labeled nucleic acids, Molecular Probes and
its distributors exclusively supply many unique and important reagents and kits. The
superior properties of our proprietary dyes ensure that the labeled nucleic acids are the
best that can be prepared by each method. Our available technologies include:
• ChromaTide dUTP, ChromaTide OBEA-dCTP 1 and ChromaTide UTP nucleotides,
which provide researchers with a large selection of fluorophore- and hapten-labeled
nucleotides that can be enzymatically incorporated into DNA or RNA probes for
FISH (fluorescence in situ hybridization), DNA arrays and microarrays and other
hybridization techniques (see Legal Notice for ChromaTide UTP and dUTP Nucleotides).
• ULYSIS Nucleic Acid Labeling Kits, which employ a fast, simple and reliable chemical method for labeling nucleic acids without enzymatic incorporation.
• ARES DNA Labeling Kits, which employ a versatile, two-step method for labeling
DNA with fluorescent dyes to achieve a uniformity and consistency of labeling that is
difficult to obtain with conventional enzymatic incorporation of labeled nucleotides.
• Alexa Fluor Oligonucleotide Amine Labeling Kits, which use familiar chemical labeling of amine-terminated oligonucleotides to prepare the best singly labeled fluorescent
conjugates.
Unit Size
Legal Notice for
ChromaTide UTP and dUTP
Nucleotides
For Research Use Only. These NEN-brand
products are distributed and sold under an
agreement between Enzo Diagnostics, Inc., and
PerkinElmer Life Sciences, Inc. (formerly NEN
Life Science Products, Inc.), for research
purposes only by the end-user in the research
market and are not intended for diagnostic or
therapeutic use. Purchase does not include or
carry any right or license to use, develop or
otherwise exploit this product commercially.
Any commercial use, development or exploitation of this product without the express prior
written authorization of Enzo Diagnostics, Inc.,
and PerkinElmer Life Sciences, Inc., is strictly
prohibited. This product or the use of this
product may be covered by one or more Enzo
patents, including the following:
US 4,707,440; US 4,952,685; US 5,002,885;
US 5,013,831; US 5,328,824; US 5,449,767;
and DK 164 407 8; and by one or more PerkinElmer patents, including US 5,047,519; US
5,151,507; and US 5,608,063.
Custom conjugations of some of our proprietary dyes to oligonucleotides for personal
research use are available from several authorized sources. A variety of additional methods for preparing labeled oligonucleotides and nucleic acids and using them in nucleic
acid sequencing are described in this section. Section 8.5 describes use of labeled nucleic
acids as hybridization reagents for microarrays, FISH and real-time PCR assays. Section
8.5 also includes a discussion of our important ELF and TSA technology for amplifying
FISH signals.
Section 8.2
287